Early recognition of pathogens and genetic diseases, and susceptibility and/or predisposition thereto is vitally important in healthcare and, at least in part, depends on the ability to detect nucleic acids with accuracy and sensitivity. Not surprisingly, DNA and RNA detection methods are now routinely used for forensic, paternity, military, environmental and other testing applications. Although some highly sensitive technologies for direct nucleic acid detection are currently under development, amplification of targeted sequences is an important component of many DNA detection systems today. Most sensitive and accurate methods are based on oligonucleotide probe detection. An oligonucleotide sequence can be chosen to form a perfect match duplex with any predetermined site of an amplified polynucleotide sequence of interest. This complementary duplex can then be detected indicating the presence of the targeted nucleic acid in the reaction mixture. In many methods based on nucleic acids amplification, the oligonucleotide probes do not need to be longer than 6-10-mers to provide for explicit detection of polynucleotide sequences of interest. Probes of this length are sequence-specific within the context of amplicons that are 100-500 base pairs long. Use of the short probes improves many aspects of the detection process and can be particularly effective, for example, in enhancing the detection signal while reducing the signal background and unambiguously identifying target polymorphic variations as small as single nucleotide polymorphism. Moreover, the 10-mer and shorter oligonucleotide probes provide opportunity to establish a complete probe inventory, a universal probe library which would always contain a complementary probe for detection of any given sequence of any target nucleic acid.
However, the probe-target duplex needs to be stable enough to enable the detection by a particular method. Generally, duplex stability depends on the duplex length and base pair composition. Detection of A/T-rich sequences is notoriously difficult due to the thermodynamic instability of A-T base pair relative to the G-C pair. Moreover, many currently used detection methods, for example, those based on the real time polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis K. B., 1987), are performed at temperatures exceeding 50-60° C. In these aspects, the probes commonly needs to be 18-20-mers or longer oligonucleotides in order to address both, the natural sequence variety of nucleic acids and the elevated temperatures of the detection methods used. A number of technologies have been proposed to enhance the oligonucleotides hybridization properties. The examples include base-modified (Lebedev Y. et al, 1996) and sugar-modified nucleotide analogs like LNA (Wengel J., Nielsen P., 2003) and PNA (Egholm M. et al, 1993), duplex-stabilizing tails like minor groove binders (Kutyavin I. V. et al, 1998) and intercalators (e.g., Asseline U. et al, 1984). With regard to the PCR-based methods, use of these chemical modifications allows to reduce the probe average length to ˜12-18-mer oligonucleotides. However, this length range is still out of reach of a reasonably-sized universal library of the detection probes.
There is therefore, a pronounced need in the art for more efficient and versatile methods of nucleic acids detection that can employ very short oligonucleotide probes, particularly shorter than <8-mers, regardless of the nucleotide composition of the detected nucleic acids of interest.